How the immune system evolves antibodies (Ab) specific for virtually any foreign molecule or antigen, (Ag), without binding any self-molecules is central to our understanding of immunology and autoimmunity. Our fundamental hypothesis is that affinity maturation contributes to this remarkable feat of molecular recognition by evolving polyspecific Ab that recognize different targets with an induced-fit mechanism into specific antibodies that recognize only their target with a lock-and-key mechanism. This suggests that germline Ab must be flexible and conformationally heterogeneous while their mature counterparts must be more rigid and conformationally homogeneous. Thus, directly testing the hypothesis requires the characterization of protein flexibility and heterogeneity as a function of affinity maturation. This has been challenging in the past, but modern experimental and computational methodologies have recently made it feasible. In fact, using 3-pulse photon echo peak shift and dynamic Stokes shift spectroscopy, we have already demonstrated that affinity maturation of anti-fluorescein Ab, Ab 4-4-20, significantly rigidified the Ab-Ag complex and dramatically reduced its conformational heterogeneity. Based on these results, we propose to use a combination of biophysical techniques (NMR, laser spectroscopy, X-ray crystallography, isothermal titration calorimetry, surface plasmon resonance, and molecular dynamics simulations) to rigorously test our fundamental hypothesis for Ab 4-4-20. We also propose to test the generality of our hypothesis with other anti-fluorescein Abs, Abs that bind another small- molecule model antigen, and importantly, and anti-HIV Ab. The Specific Aims of the proposal are; Aim 1: Determine whether 4-4-20 evolved from a flexible germline Ab that binds fluorescein via an induced- fit mechanism into a rigid mature Ab that binds fluorescein via a lock-and-key mechanism. Aim 2: Test the generality of our fundamental hypothesis with other anti-fluorescein Abs and with Abs that evolved to bind the model antigen 8-methoxypyrene-1,3,6,trisulfonic acid. Aim 3: Test the generality of our fundamental hypothesis with an anti-HIV Ab, 0.5[unreadable]. Aim 4: Generate comprehensive models of Ab 4-4-20 and 0.5[unreadable] evolution. These studies will provide the first complete characterization of any protein over all biologically relevant timescales, test our fundamental hypothesis, its generality and its physiological relevance, and reveal the molecular details of how the immune system tailors Abs for biological function.